Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system

Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.